Cartilage regeneration and related diseases like Osteoarthritis constitute an unmet medical need, with the treatments being limited to pain management or eventual total joint replacement. In order to unlock the potential of stem cells for cartilage regeneration, it is important to understand the fundamental processes governing stem cell differentiation. Epigenetic regulators like the DNA methylation marks are known to play key roles in cellular differentiation; however their role in chondrocyte differentiation is poorly understood. Recent discoveries have led to a paradigm shift in our understanding of the DNA methylation and demethylation regulatory network with the discovery of novel oxidative modifications of methylated cytosine (5mC) i.e. 5hmC, 5caC and 5fC as well as the enzymes involved in their generation and turnover i.e. Ten-eleven-translocation (TET) enzymes and Base-excision-repair (BER) glycosylates. 5hmC has been found to be stably present in DNA as well as influence gene expression as an epigenetic mark independent of 5mC. 5hmC and TET enzymes have been identified to be critical for embryonic stem cells (ESC) differentiation. Our initial studies have uncovered a dynamic increase in global 5hmC levels during chondrocyte differentiation as well as an impairment of chondrogenic differentiation both in vitro and in vivo n the absence of TET 1 and 2. This research proposal is therefore focused on defining the role of 5hmC and the TET family of enzymes in epigenetic regulation of chondrocyte differentiation. In Aim 1, we will utilize a well-established mouse chondroprogenitor cell line to determine the effects of TET1, 2 and 3 loss-of-functions on chondrocyte differentiation. As suggested by initial data, we will test the hypothesis that the TET proteins directly regulate the Sox trio-Sox 9, 5 and 6. Finally, global gene expression analyses will be performed to identify common and distinct targets of TET1, 2 and 3 in chondrocyte differentiation. In Aim 2, we will validate our in vitro findings in mice. 5hmC dynamics during embryonic limb development will be studied in both wild-type and TET1 mutant mice. Histology, immunostaining, DNA and RNA analyses will be used to define the timing and effect of 5hmC changes on chondrogenic markers including Sox9, 5 and 6, col2a1, Runx2 and col10a1 in normal limb development and how it is impaired in the mutant mice. These studies will greatly advance the understanding of the epigenetic regulation of chondrocyte differentiation by 5hmC and TET proteins, and will set the stage for testing the therapeutic potential of these novel regulators in cartilage regeneration and Osteoarthritis.